JP5462782B2 - Biology-based catalysts for delaying plant development processes - Google Patents

Description

  The present invention relates to a method for delaying plant development comprising the step of exposing a plant or plant part to one or more bacteria or enzymes. Further provided is an apparatus for delaying the plant development process.

  Ethylene production in plants and plant parts is induced by a variety of external and stress factors, including injury, application of hormones (eg, auxin), anaerobic conditions, cooling, heat, drought and pathogen infection. Additional ethylene production is also observed during various plant development processes, including fruit or vegetable ripening, seed germination, defoliation, and flower senescence.

  Ethylene biosynthesis in plants is generally shown as an enzyme scheme involving three enzymes, commonly referred to as the “Yang cycle”, where S-adenosyl-L-methionine (SAM) synthase is methionine. Catalyzes the conversion of AdoMet to S-adenosyl-L-methionine (AdoMet), 1-aminocyclopropane-1-carboxylic acid (ACC) synthase catalyzes the conversion of AdoMet to ACC, and ACC oxidase Catalyze the conversion of ethylene and by-products to carbon dioxide and hydrogen cyanide. For example, see Srivastava (2001) Plant Growth and Development: Holmons and Environment (Academic Press, New York) for an overview of ethylene biosynthesis in plants and plant development processes regulated by ethylene.

  Previous studies have demonstrated that in climacteric fruits, ripening is induced, at least in part, by a sudden and significant increase in ethylene biosynthesis. The burst of ethylene production has been linked to the fruit ripening process of climacteric fruits, but the exact mechanism is not fully understood, especially in non-climacteric fruits. Non-climacteric fruits do not have an outbreak of ethylene production, but non-climacteric fruits respond to ethylene. In addition, the amount of ethylene synthesized and also the sensitivity of certain products to ethylene varies between fruits, vegetables, and other plant products. For example, apples exhibit a high level of ethylene production and ethylene sensitivity, while artichokes exhibit a low level of ethylene production and ethylene sensitivity. For example, postharvest. ucdavis. edu / Product / Storage / index. Please refer to Cantwell (2001) “Properties and Recommended Conditions for Storage of Fresh Fruits and Vegibles”, which is incorporated herein by reference in its entirety, on the site of html (last access date, March 6, 2007). . Fruit ripening generally results in changes in color, softening of the skin, changes in fruit sugar content and flavor. Ripening initially makes the fruit more edible and attractive, but the process ultimately leads to degradation and alteration of the fruit quality, making it unfit for consumption and greatly Leading to financial loss of commercial activity. Control of the ripening process is desirable to improve the shelf life of fruits and other agricultural products undergoing ripening and to extend the time available for transport, storage and sale.

  In addition to the surge in ethylene biosynthesis in climacteric fruits, ripening-related changes are also associated with increased respiratory rates. Heat is produced as a result of the respiration of fruits, vegetables, and other plant products, and consequently affects the shelf life and required storage conditions (eg, refrigeration) of these commodities. Plant products with fast respiration rates (for example, thistle, cut flowers, asparagus, broccoli, spinach) are stored shorter than those with low respiration rates (for example, nuts, dates, apples, citrus fruits, grapes, etc.) Exhibits life. Respiration is affected by a number of environmental factors including temperature, atmospheric composition, physical stress, light, chemical stress, radiation, water stress, growth regulators, and pathogen attack. In particular, temperature plays an important role in the respiration rate. For an overview of respiratory metabolism and recommended controlled atmospheric conditions for fruits, vegetables, and other plant products, see, eg, Kader (2001) Postharvest Culture Series No. 22A: 29-70 (University of California-Davis); usna. usda. gov / hb66 / 019 respiration. Saltwater (University of California-Davis) “Respiratory Metabolism” and postharvest. ucdavis. edu / Product / Storage / index. Please refer to Cantwell (2001) “Properties and Recommended Conditions for Storage of Fresh Fruits and Vegitables” of shml (Last Access Date, March 6, 2007), which is incorporated herein by reference in its entirety. It is.

  Methods and compositions for retarding the fruit ripening process include, for example, silver salts (eg, silver thiosulfate), 2,5-norbornadiene, potassium permanganate, 1-methylcyclopropane (1-MCP), cyclopropane Including application of (CP), and derivatives thereof. These compounds have significant disadvantages, such as the presence of heavy metals, malodors, and explosive properties when compressed, making them unacceptable or limited in applicability in the food industry . Control ethylene production to delay plant development processes (eg fruit ripening) by introducing nucleic acid sequences that limit ethylene production, in particular by reducing the expression of ACC synthase or ACC oxidase enzyme A genetic recombination technique for this purpose is also under investigation. However, the public response to genetically modified agricultural products is not all favorable.

  Thus, there remains a significant need for techniques for safe methods and devices for delaying the plant development process. Such methods and apparatus provide better control of fruit ripening, vegetable ripening, flower senescence, leaf litter, and seed germination, and for various agricultural products (eg, fruits, vegetables, and cut flowers) The shelf life can be extended, thereby enabling longer transport distances for these products without the need for refrigeration, increasing the desirability of the products to consumers, and premature maturation and Reduce the financial costs associated with product loss due to aging.

  Methods are provided for delaying the plant development process including, but not limited to, fruit ripening, vegetable ripening, flower senescence, and litter. The methods of the invention generally include exposing a plant or plant part to an amount of one or more bacteria sufficient to delay the plant development process of interest. In a particular embodiment of the present invention, the bacteria are selected from the group consisting of Rhodococcus species, Pseudomonas-chlororaffith, Brevibacterium-ketoglutamicum, and mixtures thereof. Bacteria used in the performance of the method are further treated with an inducer, including, for example, asparagine, glutamine, cobalt, urea, and mixtures thereof to induce the ability of the bacteria to delay the plant development process of interest. can do.

  The present invention provides an apparatus for delaying the plant development process comprising a catalyst comprising one or more of the bacteria, in particular Rhodococcus sp., Pseudomonas chlororafis, Brevibacterium ketoglutamicum, or mixtures thereof. Any device that allows exposure of a plant or plant part to a catalyst and delays the plant development process of interest is encompassed by the present invention. Exemplary devices include those in which a catalyst is fixed in a matrix and placed in, placed on, or attached to any physical structure. In the following, various configurations of the disclosed apparatus are assumed and described in more detail. The method and apparatus of the present invention for delaying the plant development process extends the shelf life and facilitates long-distance transport of plant products such as fruits, vegetables and flowers, improves the satisfaction of consumer products, and It finds specific uses, such as reducing product loss caused by premature maturation or aging.

  Having generally described the invention as described above, reference will now be made to the accompanying drawings. They are not necessarily drawn to scale.

FIG. 5 shows a non-limiting depiction of a three layer device for delaying fruit ripening. The outer layer (shown as A and B) provides structural integrity to the device. The catalyst layer contains one or more of the enzymes of the present invention and is positioned between the outer layers, as defined below. 2A-2C provide a non-limiting depiction of various devices for delaying fruit ripening. These devices comprise a catalyst layer, one or more layers intended to provide structural integrity, and one or more layers intended to be removed prior to use of the device. Removal of one or more of these layers may, for example, expose an adhesive for attachment of the device to other physical structures. 3A-3B are non-limiting depictions of an apparatus for delaying fruit ripening. The apparatus includes a catalyst fixed on the film layer and attached to a physical structure (eg, a box suitable for fruit storage / transport). FIG. 6 provides a non-limiting depiction of an apparatus for delaying fruit ripening. The apparatus includes a slotted chamber structure that allows insertion and replacement of one or more catalyst module elements as defined below. The outer layer of the physical structure can be composed of a material that allows air flow into the catalyst.

  The invention will now be described more fully with reference to specific embodiments of the invention and, in particular, with reference to the various drawings provided therewith. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

  Throughout this specification, “comprising” or grammatical variations thereof includes a specified element, integer or step, or group of elements, multiple integers or steps, but any other element, integer or It will be understood that no step, or group of elements, multiple integers, or exclusion of steps is implied.

  The present invention provides a method for delaying a plant development process comprising exposing a plant or plant part to one or more bacteria. In certain embodiments, the method comprises exposing the plant or plant part to one or more bacteria selected from the group consisting of Rhodococcus spp., Pseudomonas chlororaphis, Brevibacterium ketoglutamicum, and mixtures thereof. A method of delaying the plant development process is derived, and one or more bacteria are exposed to the plant or plant part in an amount sufficient to delay the plant development process. Further provided is an apparatus for delaying the plant development process of interest and for performing the methods described herein. The methods and apparatus of the present invention can be used, for example, to delay fruit / vegetable ripening or flower senescence and to extend the shelf life of fruits, vegetables, or flowers, thereby Facilitates the transport, delivery and sale of fresh plant products.

  As used herein, “plant” or “plant part” refers to intact plants and fruits, vegetables, flowers, seeds, leaves, tree nuts, embryos, pollen, ovules, branches, berries, ears, cob, hulls Broadly defined to include any plant part, including, but not limited to, stems, roots, root tips, cocoons and the like. In certain embodiments, the plant part is a fruit, vegetable, or flower. In certain aspects of the invention, as detailed below, the plant part is a fruit, more specifically a climacteric fruit.

  The methods and apparatus of the present invention are aimed at delaying plant growth processes, such as those generally associated with increased ethylene biosynthesis. “Plant development process” is intended to mean any growth or development process of a plant or plant part, including but not limited to fruit ripening, vegetable ripening, flower senescence, defoliation, seed germination, etc. Is. In certain embodiments, the plant development process of interest is fruit or vegetable ripening, flower senescence, or leaf litter, more particularly fruit or vegetable ripening. “Delaying plant development process” and grammatical variations thereof, as defined herein, relate to any slowing, interruption, suppression or inhibition of the plant development process of interest, or generally related to a particular plant development process. Refers to a phenotypic or genotypic change to a plant or plant part. For example, when the plant development process of interest is fruit ripening, delayed fruit ripening generally results in ripening processes (eg, color change, pericarp (eg, ovary wall) softening, sugar, as described above) Inhibition of changes associated with increased content, changes in flavor, general deterioration / degeneration of fruits, and reduced desirability of fruits for consumers. One skilled in the art will recognize that the length of time required for a fruit to ripen will vary depending on, for example, the type of fruit and the particular storage conditions utilized (eg, temperature, humidity, airflow, etc.) You will recognize. Thus, “retarding fruit ripening” is a delay of 1 to 90 days, especially 1 to 30 days, more specifically 5 to 30 days. Methods for assessing delays in plant development processes such as fruit ripening, vegetable ripening, flower senescence, and defoliation are well within the routine ability of those skilled in the art, e.g., untreated plants or plant parts Based on comparison with plant growth process in In certain aspects of the invention, the delay in the plant development process caused by the performance of the method is with an untreated plant or plant part, or with one or more agents known to delay the plant development process of interest. It can be evaluated relative to the treated plant or plant part. For example, the delay in fruit ripening resulting from the practice of the method of the invention can be compared to the fruit ripening time of untreated fruit or fruit treated with an anti-maturation agent as described above.

The methods of the invention for delaying the plant development process generally comprise exposing a plant or plant part to one or more of the following bacteria: Rhodococcus spp., Pseudomonas chlororafis, Brevibacterium ketoglutamicum, or a mixture comprising any combination of these bacteria. In certain embodiments, the one or more bacteria comprises Rhodococcus spp., More specifically Rhodococcus sp. Strain DAP 96253, Rhodococcus rhodochrous DAP 96622 strain, Rhodococcus erythropolis, or mixtures thereof. As used herein, exposure of a plant or plant part to one or more of the above-mentioned bacteria can include, for example, intact bacterial cells, bacterial cell lysates, and bacterial extracts with enzymatic activity (ie, “ Exposure to the enzyme extract "). Methods for preparing lysates and enzyme extracts from cells, including bacterial cells, are routine in the prior art. One or more bacteria used in the methods and apparatus of the present invention may be more generally referred to herein as “catalysts”.

  According to the method of the present invention, one or more bacteria are exposed to a plant or plant part in an amount sufficient to delay the plant development process. “Exposing” a plant or plant part to one or more of the bacteria of the invention includes any method for providing bacteria to the plant or plant part. Indirect exposure methods include, for example, placing a bacterium or mixture of bacteria on a plant or plant part in general proximity (ie, indirect exposure). In other embodiments, the bacteria may be exposed closer to the plant or plant part, or through direct contact. In addition, as defined herein, a “sufficient” amount of one or more bacteria of the present invention may be determined by the specific bacteria utilized in the method, such as intact, as described above. Including, but not limited to, forms exposed to plants or plant parts (as bacterial cells, cell lysates, or enzyme extracts), methods by which bacteria are exposed to plants or plant parts, and length of exposure time, Depends on various factors. It will be routine for those skilled in the art to determine the “sufficient” amount of one or more bacteria necessary to delay the plant development process of interest.

  In certain embodiments of the invention, the one or more bacteria are selected from the group consisting of Rhodococcus spp., Pseudomonas chlororafis, Brevibacterium ketoglutamicum, and mixtures thereof, but are exposed to the plant or plant part. Any bacteria that delays the plant development process can be used in the method and apparatus. For example, the genus Nocardia [see Japanese Patent Application No. 54-129190], Rhodococcus [see Japanese Patent Application No. 2-470], Rhizobium [see Japanese Patent Application No. 5-236777] Klebsiella [Japan Patent Application No. 5-30982], Aeromonas [Japan Patent Application No. 5-30893], Agrobacterium [Japan Patent Application No. 8-154691], Bacillus [Japan Patent] Application No. 8-187092], Pseudocardia [Japanese Patent Application No. 8-56684], Pseudomonas, and bacteria belonging to Mycobacterium are non-limiting examples of microorganisms that can be used according to the present invention. . Not all species within a given genus can exhibit the same characteristics. Thus, it generally includes one or more species that include a strain that can exhibit a desired activity (eg, the ability to delay certain plant development processes such as fruit ripening) but generally does not exhibit the desired activity. It is possible to have a known genus. However, in light of the disclosure provided herein and general knowledge of the prior art, one of ordinary skill in the art would perform an assay to determine whether a particular species has one or more of the desired activities. It will be a routine experiment.

Specific examples of bacteria useful according to the invention include Nocardia species, Rhodococcus species, Rhodococcus rhodochrous, Klebsiella species, Aeromonas species, Citrobacter freundi, Agrobacterium rhizonegues, Agrobacterium tumefaciens, Xantobacter -Flabus, Erwinia-niglifluens, Enterobacter species, Streptomyces species, Rhizobium species, Rhizobium-roti, Rhizobium-Legminosarum, Rhizobium-Merioti, Candida-Gilliammondi, Pantoea-Agromerans, Klebsiella-Pneumoniae subspecies pneumoniae Um-Radiobacter, Bacillus-Smithii, Pseudocardia-Thermophylla, Pseudomonas-Chlorophyllus, Pseudomonas Erythropolis, Brevibacterium - ketoglutamicum, Rhodococcus - erythropolis, Nocardia - Farushinika, Pseudomonas - aeruginosa, and Helicobacter - but pylori include, but are not limited to. In certain embodiments, bacteria from the genus Rhodococcus, more specifically Rhodococcus sp. Strain DAP 96253 (ATCC deposit no. 55899; deposited with ATCC on December 11, 1996), Rhodococcus rhodochrous DAP 96622 strain (ATCC deposit) No. 55898; deposited at ATCC on Dec. 11, 1996), Rhodococcus erythropolis, or mixtures thereof are used in the methods and apparatus of the present invention.

  In certain embodiments of the invention, one or more bacteria may exhibit desired characteristics (eg, the ability to delay plant development processes such as fruit ripening) upon exposure to or treatment with a suitable inducer. "Induced" to. Inducing agents include, but are not limited to, asparagine, glutamine, cobalt, urea or any mixture thereof. In certain embodiments, the bacterium is exposed to or treated with an asparagine inducer, more specifically a mixture of inducers comprising asparagine, cobalt, and urea. The inducer can be added at any time during the cultivation of the desired cells. For example, for bacteria, the medium can be supplemented with an inducing agent prior to initiating bacterial culture. Alternatively, the bacteria can be cultured on the medium for a predetermined period of time to allow the bacteria to grow, and the inducing agent is added one or more predetermined times to induce the desired enzyme activity in the bacteria. can do. Furthermore, the inducing agent can be added to the growth medium (or to a separate mixture containing pre-grown bacteria) so as to induce the desired activity of the bacteria after the growth of the bacteria is complete.

  While not intending to be limited to a particular mechanism, the step of “inducing” the bacterium of the present invention is the production (or increased production) of one or more enzymes such as nitrile hydratase, amidase, and / or asparaginase. And induction of one or more of these enzymes may serve to delay the plant development process of interest. “Nitrile hydratase”, “amidase”, and “asparaginase” include a group of enzymes present in cells from various organisms, including but not limited to bacteria, fungi, plants, and animals. Such enzymes are well known to those skilled in the art and each enzyme has a recognized enzymatic activity. “Enzyme activity”, as used herein, generally refers to the ability of an enzyme to function as a catalyst in a process, such as converting one compound to another. Specifically, nitrile hydratase catalyzes the hydrolysis of nitrile (or cyanohydrin) to the corresponding amide (or hydroxy acid). Amidases catalyze the hydrolysis of amides to the corresponding acids or hydroxy acids. Similarly, asparaginase enzymes such as asparaginase I catalyze the hydrolysis of asparagine to aspartic acid.

  In certain embodiments of the invention, enzyme activity can be expressed in terms of “units” per mass of enzyme or cell (generally based on cell dry weight, eg, units / mg cdw). “Unit” generally refers to the ability to convert a particular amount of a compound into a different compound under a specified set of conditions as a function of time. In certain embodiments, one “unit” of nitrile hydratase activity corresponds to 1 μmol of acrylonitrile per milligram (dry weight) of cells per minute at a pH of 7.0 and a temperature of 30 ° C. It can be related to the ability to convert to an amide. Similarly, one unit of amidase activity relates to the ability to convert 1 μmol of acrylamide to its corresponding acid per milligram of cells (dry weight) per minute at a pH of 7.0 and a temperature of 30 ° C. be able to. Furthermore, one unit of asparaginase activity can be related to the ability to convert 1 μmol of asparagine to its corresponding acid per milligram of cells (dry weight) per minute at pH 7.0 and temperature of 30 ° C. . Assays for measuring nitrile hydratase, amidase activity, or asparaginase activity are known in the art and include, for example, detection of free ammonia. Faecett and Scott (1960). Clin. Pathol. 13: 156-159, which is hereby incorporated by reference in its entirety.

  A method of delaying a plant development process comprising exposing a plant or plant part to one or more enzymes selected from the group consisting of nitrile hydratase, amidase, asparaginase, or mixtures thereof, comprising: Further encompassed by the present invention is a method in which an amount or enzyme activity level of one or more enzymes sufficient to delay the process is exposed to the plant or plant part. For example, the entire cell that produces, is induced to produce, or has been genetically modified to produce one or more of the above-described enzymes (ie, nitrile hydratase, amidase, and / or asparaginase) It can be used in a method of delaying the plant development process. Alternatively, nitrile hydratase, amidase, and / or asparaginase can be isolated, purified, or semi-purified from any of the cells described above and exposed to the plant or plant part in a more isolated form. . For example, Goba et al. (2001) J. Org. Biol. Chem. 276: 23480-23485; Nagasawa et al. (2000) Eur. J. et al. Biochem. 267: 138-144; Soong et al. (2000) Appl. Environ. Microbiol. 66: 1947-1952; Kato et al. (1999) Eur. J. et al. Biochem. 263: 662-670, all of which are hereby incorporated by reference in their entirety. One skilled in the art will further recognize that a single cell type can produce (or induce, or recombine genes) to produce more than one enzyme of the invention. Let's go. Such cells are suitable for use in the disclosed methods and devices.

  Nucleotide and amino acid sequences of several nitrile hydratases, amidases, and asparaginases obtained from various organisms are disclosed in publicly available sequence databases. A non-limiting list of representative nitrile hydratases and aliphatic amidases known in the art is set forth in Tables 1 and 2 and the Sequence Listing. The “protein score” for Tables 1 and 2 provides an overview of the percentage of confidence intervals (% confidence intervals) in isolated protein identification based on mass spectroscopic data.

  In general, any bacterial, fungal, or animal cell that can produce or be induced to produce nitrile hydratase, amidase, asparaginase, or any combination thereof can be used in the practice of the invention. Nitrile hydratase, amidase, and / or asparaginase can be produced constitutively in a cell from a particular organism (eg, a bacterium, fungus, plant cell, or animal cell), or alternatively, the cell can be Only “induction” with a suitable inducer can produce the desired single or multiple enzymes. “Constitutively” is intended to mean that at least one enzyme of the invention is continuously produced or expressed in a particular cell type. However, other cell types are “induced” to express nitrile hydratase, amidase, and / or asparaginase in an amount or cellular activity level sufficient to delay the plant development process of interest, as described above. May need to be done. That is, the enzymes of the present invention can only be produced (or produced at sufficient levels) after exposure to or treatment with a suitable inducer. Such inducers are known in the art and have been outlined above. For example, in certain embodiments of the invention, one or more bacteria are treated with an inducing agent such as asparagine, glutamine, cobalt, urea, or any mixture thereof, more specifically a mixture of asparagine, cobalt, and urea. Is done. In addition, asparaginase I activity is disclosed in pending US patent application Ser. No. 11 / 669,011, filed Jan. 30, 2007, entitled “Induction and Stabilization of Enzymatic Activity in Microorganisms”. Can be induced in Rhodococcus rhodochrous DAP 96622 (Gram positive) or Rhodococcus sp. DAP 96253 (Gram positive) in a medium supplemented with amides containing amino acids, or derivatives thereof. Similarly, other Rhodococcus strains can be selectively induced to exhibit asparaginase I enzyme activity utilizing amides containing amino acids or derivatives thereof.

  In another embodiment of the present invention, P. chlororafis (ATCC Deposit No. 43051) that produces asparaginase I activity in the presence of asparagine, and a Gram-positive bacterium that also showed production of asparaginase activity, Kletoglutamicum (ATCC deposit number 21533) is used in the disclosed method. Fungal cells expressing nitrile hydratase, amidase, and / or asparaginase, such as those from Fusarium, plant cells, and animal cells, are also isolated as a whole cell or one or more of the above-mentioned enzymes. Can be used in the methods and apparatus disclosed herein.

  In further embodiments, host cells that have been genetically modified to express nitrile hydratase, amidase, and / or asparaginase are exposed to plants or plant parts according to the present methods and apparatus for delaying plant development processes. Can be used for Specifically, a polynucleotide encoding nitrile hydratase, amidase, or asparaginase (or a complex polynucleotide, each encoding nitrile hydratase, amidase, or asparaginase) is selected from the enzymes of the present invention. Can be induced in a host cell by standard molecular biology techniques to produce genetically modified cells that express one or more of The use of the terms “polynucleotide”, “polynucleotide construct”, “nucleotide” or “nucleotide construct” is not intended to limit the invention to polynucleotides or nucleotides comprising DNA. One skilled in the art will recognize that polynucleotides and nucleotides can include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both natural molecules and synthetic analogs. The polynucleotides of the present invention include all sequence forms including, but not limited to, single-stranded forms, double-stranded forms, and the like.

  Polynucleotide variants and fragments encoding polypeptides that maintain the desired enzymatic activity (ie, nitrile hydratase, amidase, or asparaginase activity) can also be used in the practice of the invention. A “fragment” is intended to be a portion of a polynucleotide and therefore also encodes a portion of the corresponding protein. Polynucleotides that are fragments of enzyme nucleotide sequences are generally at least 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, It includes 800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400 contiguous nucleotides, or the number of nucleotides present in the enzyme polynucleotide sequence up to full length. A polynucleotide fragment encodes a polypeptide having the desired enzymatic activity and is generally at least 15, 25, 30, 50, 100, 150, 200, or 250 contiguous amino acids, or up to the full length of the invention. Encode up to the total number of amino acids present in the enzyme amino acids. “Variants” are intended to mean substantially similar sequences. In general, variants of a particular enzyme sequence of the invention are at least about 40%, 45%, 50%, 55%, 60%, 65 relative to a reference enzyme sequence, as determined by standard sequence alignment programs. %, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity Have sex. Variant polynucleotides encompassed by the present invention encode polypeptides having the desired enzyme activity.

  When used in reference to the production of genetically modified cells, the term “introducing” refers to providing a polynucleotide encoding a nitrile hydratase, amidase and / or asparaginase to a host cell, particularly a microorganism such as E. coli. Intended to mean. In some embodiments, the polynucleotide is provided in such a way that the sequence utilizes the interior of the host cell, including its potential insertion into the genome of the host cell. The method of the invention does not rely on a particular method for introducing the sequence into the host cell, only the polynucleotide is accessible inside the at least one host cell. Methods for introducing polynucleotides into host cells are well known in the art and include, but are not limited to, stable transfection methods, transient transfection methods, and virus-mediated methods. . “Stable transfection” is intended to mean that a polynucleotide construct introduced into a host cell can be integrated into the genome of the host and inherited by its progeny. “Transient transfection” or “transient expression” is intended to mean that the polynucleotide is introduced into the host cell but is not integrated into the genome of the host.

  In addition, nitrile hydratase, amidase, or asparaginase nucleotide sequences can be included, for example, on a plasmid for introduction into a host cell. Exemplary plasmids of interest include vectors having defined cloning sites, origins of replication, and selectable labels. The plasmid can further comprise transcription and translation initiation sequences and transcription and translation terminators. A plasmid also includes a gene containing at least one independent terminator sequence, a sequence that allows for replication of the cassette in eukaryotes, prokaryotes, or both, and a selection marker for both prokaryotic and eukaryotic systems. An expression cassette can also be included. Vectors are suitable for the replication and integration of prokaryotes, eukaryotes, or optimally both. For an overview of cloning, packaging, and expression systems and methods, see Giliman and Smith (1979) Gene 8: 81-97; Roberts et al. (1987) Nature 328: 731-734; Berger and Kimmel (1989) Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152 (Academic Press, Inc., San Diego, California); Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Vols. 1-3 (2d ed; Cold Spring Harbor Laboratory Press, Plainview, New York); and Ausbel et al. , Eds. (1994) Current Protocols in Molecular Biology, Current Protocols (see Greene Publishing Associates, Inc., and John Wiley & Sons, Inc., New York; supplemented in 1994). Genetically modified host cells that express one or more of the enzymes of the invention can be used as a whole cell or as a biological source from which one or more enzymes of the invention can be isolated and the disclosed methods and Can be used in the device.

Further provided is an apparatus for delaying the plant development process and for carrying out the method of the present invention. In certain embodiments, plant growth processes, particularly fruit, comprising a catalyst comprising one or more bacteria selected from the group consisting of Rhodococcus spp., Pseudomonas chlororafis, Brevibacterium ketoglutamicum, and mixtures thereof. Devices for delaying maturity are encompassed by the present invention. Rhodococcus sp. DAP 96253, Rhodococcus rhodochrous DAP 96622, Rhodococcus erythropolis, or mixtures thereof can be used in certain embodiments of the invention. One or more bacteria of the device of the invention are provided in an amount sufficient to delay the plant development process, as defined above. In other embodiments of the invention, the catalyst comprises one or more enzymes (ie, nitrile hydratase, amidase, and / or asparaginase) in an amount or level of enzyme activity sufficient to retard the plant development process. The desired enzyme source for use as a catalyst in the apparatus of the present invention is also detailed above. For example, the catalyst can be used in the form of whole cells, producing (or induced to produce, or genetically modified) one or more of the enzymes of the invention, or isolated, purified. The enzyme itself can be included in semi-purified form.

  The apparatus for delaying the plant development process encompassed by the present invention can be provided in a variety of suitable forms and is suitable for single use or multiple uses (eg, “rechargeable”). possible. Furthermore, the device of the present invention finds use in both residential and commercial environments. For example, such devices can be integrated into residential or commercial refrigerators, included in trains, trucks, etc., for long distance transport of fruits, vegetables, or flowers, or such plant products Can be used as a stand-alone cabinet for storage or transport. An exemplary, non-limiting apparatus of the present invention is shown below and in FIGS.

  In certain embodiments, the catalyst is provided in an immobilized format. Any process or matrix for immobilizing the catalyst can be used as long as the ability of one or more bacteria (or enzymes) to delay the plant development process is maintained. For example, the catalyst can be immobilized in a matrix comprising alginic acid (eg, calcium alginate), carrageen, DEAE cellulose, or polyacrylamide. Other such matrices are well known in the art and further include any suitable cross-linking, including but not limited to glutaraldehyde or polyethyleneimine, to increase the mechanical strength of the catalyst matrix. It can be crosslinked with an agent. In one aspect of the invention, the catalyst is immobilized within a glutaraldehyde crosslinked DEAE cellulose matrix. The catalyst, in particular an immobilized form of the catalyst, may further be provided as a “catalyst module element”. The catalyst module element comprises a catalyst, such as an immobilized catalyst, in a further structure, for example, reducing potential contact with the catalyst, facilitating catalyst exchange, or allowing air flow across the catalyst. .

  In one embodiment, the matrix comprises alginic acid or a salt thereof. Alginate is a homopolymer of (1-4) linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) residues, which are covalently linked together in different sequences or blocks, respectively. And a linear copolymer. Monomers can be found in consecutive G residues (G blocks), consecutive M residues (M blocks), homopolymer blocks of alternating M and G residues (MG blocks), or randomly organized blocks it can. In one embodiment, calcium alginate is used as a substrate, more specifically as calcium alginate crosslinked with polyethyleneimine or the like to form a reinforced calcium alginate substrate. A description of such immobilization techniques can be found in Bucke (1987) “Cell Immobilization in Calcium Alginates” in Methods in Enzymology, Vol. 135 (B) (Academic Press, Inc., San Diego, California; Mosbach, ed.), Which is incorporated herein by reference. An exemplary immobilization method using polyethyleneimine cross-linked calcium alginate is also described in Example 5 below. In another embodiment, the matrix comprises an amide containing polymer. Any polymer that contains one or more amide groups can be used in accordance with the present invention. In one embodiment, the substrate comprises a polyacrylamide polymer.

  An increase in the mechanical strength of the immobilized catalyst matrix can be achieved through cross-linking. For example, cells can be chemically cross-linked to form cell aggregates. In one embodiment, harvested cells are cross-linked using glutaraldehyde. For example, the cells can be suspended in a mixture of deionized water and glutaraldehyde, after which polyethyleneimine is added until maximum aggregation is achieved. Cross-linked cells (generally in the form of particles formed by a large number of cells) can be collected by simple filtration. A further description of such techniques can be found in Lopez-Gallego et al. (2005) J. Org. Biotechnol. 119: 70-75, which is incorporated herein by reference in its entirety. A general protocol for immobilization of cells, particularly Rhodococcus sp. Cells, in DEAE cellulose crosslinked with glutaraldehyde is also outlined in Example 4 below.

  In certain aspects of the invention, the immobilized catalyst or one or more catalyst module elements are disposed within, on or attached to a “physical structure”. Physical structures include, but are not limited to, films, sheets, covering layers, boxes, pouches, bags, and slotted chambers that can hold one or more catalyst module elements. In certain embodiments, the physical structure comprises a container suitable for the transport or storage of fruits, vegetables, or flowers. The physical structure may further comprise two or more individual structures, whereby all of the individual structures are connected to a central catalyst or catalyst module element. The physical structures described herein above may optionally be refrigerated by external means, or may include a refrigeration unit within the physical structure itself.

Monitor the effectiveness of the catalyst to delay the plant development process of interest (determine when to replace the catalyst or catalyst module), or measure airflow, moisture / humidity, and carbon dioxide levels An element for monitoring may optionally be included in the apparatus of the present invention. Any device for delaying the plant development process may further comprise one or more elements to allow air flow to or through the catalyst or catalyst module element. Those skilled in the art will readily envision other possible variations to the apparatus described herein for monitoring and controlling the atmospheric conditions of a catalyst, catalyst module element, or physical structure. Conditions such as temperature, atmospheric composition (eg, relative humidity, O ₂ and CO ₂ levels), physical stress, light, chemical stress, radiation, water stress, growth regulators, and pathogen attack are important in respiratory rate Play a major role and greatly affect the shelf life of fruits, vegetables, flowers, and other plant-related products. Although the temperature and atmospheric conditions for storage will vary depending on the fruit, vegetable, or plant product of interest, the recommended storage temperature is generally in the range of about 0 ° C. to 20 ° C. and O ₂ and CO ₂ levels, respectively, in the range of about 1-10% and 0-20%. For storage of fruits, vegetables and related plant products, a relative humidity of about 50% to about 100% is generally recommended, in particular 85% to 95%, more specifically about 90% to 95%. . In view of the important correlation between the respiration rate of plant products and the shelf life, control of the above factors is important in order to delay such product alteration. Therefore, a carbon dioxide scavenger can be provided in the device so as to reduce the amount of carbon dioxide.

In certain embodiments of the invention, an air permeable catalytic device for delaying the plant development process is provided comprising a plurality of layers. For example, as shown in FIG. 1, the catalytic device 10 can include outer layers 12 and 14 and an intermediate catalyst layer 16 positioned between the outer layers 12 and 14. The catalyst layer 16 is composed of one or more bacteria (eg, Rhodococcus spp., Pseudomonas chlororafis, Brevibacterium ketoglutamicum, and mixtures thereof) or enzymes (nitrile hydratase, amidase, asparaginase, and mixtures thereof). One or more bacteria or enzymes provided in an amount sufficient to delay the plant development process of interest and a third layer. In this embodiment, one or more of the outer layers 12 and 14 provide structural integrity to the catalytic device 10. The outer layers 12 and 14 generally allow air flow to the catalyst layer 16, but in some embodiments, for example, the device forms the side of the box, and the outermost layer of the box is the catalyst layer. It may be advantageous to have a non-breathable outer layer if it is desired not to be exposed to the environment. The catalytic device 10 can be provided in a reusable or non-reusable bag or pouch according to the present invention. In one embodiment, the catalyst layer 16 comprises Rhodococcus seed cells, particularly Rhodococcus sp. Strain DAP 96253, Rhodococcus rhodochrous DAP 96622, Rhodococcus erythropolis, or mixtures thereof. Bacterial cells utilized as catalysts in the devices of the present invention can be induced by one or more inducers (eg, asparagine, glutamine, cobalt, urea, or mixtures thereof), as detailed above.

  2A-2C show an alternative device according to the present invention for delaying the plant development process. These devices include multiple layers, one or more of the layers being removable. As shown in FIG. 2A, the device can include a breathable structural layer 22 and a catalyst layer 24. Removable layers 26 and / or 28 can be provided along structural layer 22 and / or catalyst layer 24 and are generally intended to be removed prior to using or activating the catalyst. doing. In certain embodiments of the invention, removal of removable layers 26 and 28 exposes an adhesive that facilitates placement or attachment of the catalyst structure to a separate physical structure. FIG. 2B shows an alternative embodiment in which the device 30 includes two breathable structural layers 32 and 34, an intermediate catalyst layer 36, and a removable layer 38. FIG. 2C shows yet another embodiment where the device 40 includes two breathable structural layers 42 and 44, an intermediate catalyst layer 46, and two removable layers 48 and 50.

  3A-3B show an alternative embodiment 60 in which the catalyst is mounted inside a container such as a paperboard box. As shown in FIG. 3A, the container side 62 includes a catalyst layer 64 attached thereto through the use of an adhesive layer 66. A peelable film 68 can be provided adjacent to the catalyst layer 64 to protect the catalyst layer from exposure to the environment. The peelable film 68 can be removed to activate the catalyst in the catalyst layer 64 by exposing the catalyst to plant parts provided in the container, thereby delaying unwanted plant development processes. .

  FIG. 3B shows the catalyst structure 70 before the catalyst structure is mounted inside the container in the manner shown in FIG. 3A. The catalyst structure 70 includes a further peelable film 72 in addition to the catalyst layer 64, the adhesive layer 66, and the peelable film 68. The peelable film 72, like the peelable film 68, protects the catalyst structure 70 when it is packaged, shipped or stored. The peelable film 72 can be removed in the manner shown in FIG. 3A so that the pressure-sensitive adhesive layer 66 is exposed and the catalyst structure 70 can be attached inside the container.

  FIG. 4 shows a catalyst structure 80 that includes two slots 82 and 84 for receiving a catalyst cassette (eg, cassette 86). The catalyst cassette 86 is breathable and can be easily inserted into or removed from the slot 84. Thus, the catalyst cassette 86 can be easily replaced when it is desired to use a new catalyst cassette in the catalyst structure 80. The catalyst cassette 86 includes a catalyst as described herein and is preferably secured within the matrix. The catalyst structure 80 can include opposed breathable surfaces 88 and 90, such as mesh screens, to allow air flow through the catalyst cassette 86. The catalyst structure 80 may, in alternative embodiments, have only one breathable surface, two non-opposing breathable surfaces, or three or more breathable surfaces, as will be appreciated by those skilled in the art. Can be included. Although FIG. 4 includes two slots 82 and 84 for receiving a catalyst cassette (eg, cassette 86), those skilled in the art will recognize that the catalyst structure 80 has one or more slots for receiving a cassette. It will be understood that can be included. The catalyst structure 80 may be provided in a container used to transport plant parts such as fruits or flowers, or by using an adhesive layer as discussed herein, for example. Can be attached to the container.

  The method and apparatus can be used to delay the plant development process of any plant or plant part of interest. In certain embodiments, the methods and apparatus of the present invention are aimed at delaying ripening, and the plant part is a fruit (climacteric or non-climacteric), vegetable, or other that undergoes ripening It is a plant part. Those skilled in the art will appreciate that “climacteric fruits” exhibit sudden ethylene production during fruit ripening, whereas “non-climacteric fruits” generally have a significant increase in ethylene biosynthesis during the ripening process. You will recognize that you cannot expect to receive. Illustrative fruits, vegetables, and other plant products of interest include apples, apricots, biliba, breadfruit, cherimoya, feijoa, figs, guava, jackfruit, kiwi, bananas, peaches, avocados, apples, cantaloupe , Mango, muskmelon, nectarine, persimmon, support, togebanreishi, olive, papaya, passion fruit, pear, plum, tomato, pepper, blueberry, cacao, cashew, cucumber, grapefruit, lemon, lime, pepper, cherry, orange, Examples include, but are not limited to, grapes, pineapples, strawberries, watermelons, tamarillos, and nuts.

  In other aspects of the invention, methods and apparatus are provided for delaying flower senescence, wilting, organ detachment, or petal closure. Any flower may be used in the practice of the present invention. Illustrative flowers of interest include, but are not limited to, roses, carnations, orchids, purslane, usbeneer, and begonia. Of particular interest are cut flowers, and more particularly commercially important cut flowers such as roses and carnations. In certain embodiments, ethylene sensitive flowers are used in the practice of the present invention. The flowers that are sensitive to ethylene include the genus Lily, Anemone, Anthurium, Snapdragon, Chrysanthemum, Astilbe, Cattleya, Cymbidium, Dahlia, Dendrobium, Nadesico, Eustoma, Freesia, Gerbera, Examples include, but are not limited to, gypsophila, iris, lily, lily, limonium, neline, rosa, hasidy, tulip, and zinnia. Typical ethylene-sensitive flowers include Amaryllidaceae, Alliumaceae, Lilyaceae, Chrysaceae, Hyacinthaceae, Lilyaceae, Orchidaceae, Pomegranateaceae, Cactiaceae, Oleaceae, Radish, Gentianaceae, Gentianaceae Mallow, iridaceae, solanaceae, solanaceae, agaveaceae, asphodel, asperaceae, eucalyptaceae, honeysuckle, scorpionaceae, euphorbiaceae, legumes, periwinkle, myrtaceae, red-bellied, saxifrage Some of them are also included. For example, Van Doom (2002) Anals of Botany 89: 375-383; Van Doom (2002) Anals of Botany 89: 689-693, and hornet. co. nz / specifications / shortfacts / hf305004. See Elgar (1998) “Cut Flowers and Foliage- Cooling Requirements and Temperature Management” of htm (Last Access Date, March 20, 2007), which is incorporated herein by reference in its entirety. Methods and apparatus for delaying leaf fall are also encompassed by the present invention. In the plant, fruit, vegetable, and flower industries, there is great commercial interest in methods and devices for regulating plant development processes such as ripening, senescence, and organ withdrawal.

  One of ordinary skill in the art can use any of the methods or apparatus disclosed herein to increase plant growth processes, particularly generally ethylene biosynthesis (eg, fruit / vegetable ripening, flower senescence, and litter). It will be further appreciated that it can be combined with other known methods for delaying the processes involved. Furthermore, as noted above, increased ethylene production has also been observed during attack of plants or plant parts by pathogenic organisms. Thus, the methods and devices of the present invention may find further use in improving response to pathogens.

  The following examples are provided by way of illustration and are not intended to be limiting.

Experiments Hereinafter, the present invention will be described with reference to various specific examples. The following examples are not intended to limit the invention, but rather are provided as exemplary embodiments.

Example 1: Delayed Fruit Maturation by Exposure to Induced Rhodococcus Species Rhodococcus seed cells induced by asparagine, acrylonitrile, or acetonitrile were immobilized within a glutaraldehyde cross-linked matrix of DEAE cellulose. A method for inducing cells and preparing the matrix described above is described in detail below.

  The cross-linked DEAE cellulose catalyst matrix is placed in three separate paper bags (filled with about 1-2 grams of wet cells per bag) and each bag contains immature bananas, peaches, or avocados It was. As a negative control, the same fruit was placed in a separate paper bag in the absence of catalyst matrix. Paper bags were kept at room temperature and the product was observed daily for signs of fruit ripening and degradation.

  All products exposed to the catalyst matrix showed a significant delay in fruit ripening. More specifically, in the presence of the catalyst matrix, peach hardness and skin integrity were maintained longer. Similarly, for bananas, the appearance of brown spots was delayed and the hardness was maintained longer than the negative control.

Example 2: General fermentation and induction protocol The fermentation process Rhodococcus sp. Strain Rhodococcus DAP 96622 and Rhodococcus sp. DAP 96 25 3 for fermentation in other experiments include the following general Protocols and media were utilized.

  The fermentation vessel consisted of a lytic enzyme (DO) and probe for measuring pH and a sampling device for measuring glucose concentration (offline). Additional ports were used to add straighteners (eg, acids, bases, or antifoams), inducers, nutrients, and supplements. The pre-cleaned container was disinfected on site. A suitable basal medium (1 or 1.5 times) R2A or R3A was used. Specific components of these media will be described later. In some experiments, certain substitutions were made on the contents of the medium. For example, sometimes Proflo® (Trader's Protein, Memphis, TN) was used in place of proteosepeptone and / or casamino acid. Furthermore, in some experiments, instead of Proflo (Traders' Protein, Memphis, TN), Hy-Cotton 7803 (Registered Trademark) (Quest International, Hoffman Estates, IL), Cottensed Hydrates, Cottonase Ultrafiltered (Marcor Development Corp., Cartstadt, NJ) was used.

  The feed profile for nutrient supplementation was set to gradually replace the R2A or R3A basal medium with a richer medium, ie, 2X YEMEA, the components of which are also detailed below. Other optional nutrient supplements include maltose 50% (w / v) and dextrose 50% (w / v). Occasionally, products containing dextrose equivalents (glucose, maltose, and higher polysaccharides) were used in place of maltose and dextrose.

Inoculum was prepared from cultures of Rhodococcus rhodochrous DAP 96622 and Rhodococcus sp. Strain DAP 96 25 3 on suitable solid media and cultured at their appropriate temperature (eg, 30 ° C.). In certain embodiments, the cells were grown on YEMEA agar plates for 4-14 days, preferably 7 days. Alternatively, the inoculum was prepared from a frozen cell concentrate from a previous fermentation operation. Cell concentrates were generally prepared at a concentration greater than 20 times that present in the fermentor. In addition, inoculum was sometimes prepared from a suitable two-phase medium (ie, a combination of liquid media covering a solid medium of the same or different composition). When using a two-phase medium, the medium generally contained YEMEA in both the liquid and solid layers.

For induction of nitrile hydratase, CoCl ₂ -6H ₂ O and urea were added at t = 0 hours to achieve 5 to 200 ppm CoCl ₂ and 750 mg / l to 10 g / l urea, In general, 10 to 50 ppm CoCl ₂ and 7500 mg / l to 7.5 g / l urea are preferred. In one particular embodiment, urea and / or cobalt was added again during the fermentation. For example, equal volumes of urea and 150 ppm CoCl ₂ were added in 4-6 hours or in 24-30 hours. In addition to urea, addition of acrylonitrile / acetonitrile at a final concentration of 300-500 ppm, or 0.1M-0.2M asparagine was started at various times and was done stepwise or at a constant rate. When the cell mass and enzyme concentration became acceptable, the fermentation operation was terminated, typically after 24-96 hours.

  The cells were then harvested by any acceptable method, including but not limited to batch or continuous centrifugation, decanting, or filtration. Harvested cells were resuspended to a 20-fold concentrated volume in a suitable buffer such as 50 mM phosphate buffered saline (PBS) supplemented with inducers used during the fermentation process. . The cell concentrate was then frozen, in particular by flash freezing. Frozen cells were stored at −20 ° C. to −80 ° C. or under liquid nitrogen for later use.

Medium description

Guidance
The following general protocol was utilized for induction of Rhodococcus rhodochrous DAP 96622 and Rhodococcus sp. DAP 96 25 3.

  Volatile liquid inducers (eg acrylonitrile / acetonitrile) were added volumetrically as filter sterilized liquid inducers based on the concentration of the specific liquid inducer. In the case of solid inducers (eg, asparagine / glutamine), the solids were weighed and added directly to the culture medium. The resulting medium was autoclaved. When using a filter sterilized liquid inducer, the medium was autoclaved alone and cooled to 40 ° C. before adding the liquid inducer. Typical concentrations of inducers of interest were 500 ppm acrylonitrile / acetonitrile, 500 ppm asparagine / glutamine, and 50 ppm succinonitrile. The cells were then grown on the designated medium and further analyzed for specific enzyme activity and biomass.

Example 3: Nitrite Hydratase, Amidase, and Asparaginase Activity and Analysis of Biomass in Asparagine-Induced Rhodococcus Species Cells Nitrile hydratase, amidase, and asparaginase activity, and biomass were analyzed for Rhodococcus rhodochrous DAP 96622 and Rhodococcus Evaluated in asparagine-derived cells from the species DAP 96 25 3. The components of the medium, the mode of administration, the rate and the concentration of asparagine provided to the cells, as well as various variations on the cell source, were analyzed with respect to the activity of the enzymes described above and their effect on biomass. Sections A through G of this example describe the details of each set of test conditions and provide a summary of enzyme activity and biomass obtained under each specified condition.

A. Basically, as described in Example 2 above, a 20 liter fermentor inoculated with Rhodococcus sp. DAP 96253 cells taken from a solid medium is continued with the inducer asparagine. (0.2 M solution, 120 μl / min). Hy-Cotton 7803® was used in place of proteose peptone # 3 in the R3A medium described above. At the end of the fermentation run, acrylonitrile-specific nitrile hydratase activity, amidase activity, and biomass were measured according to standard techniques known in the art.

  Nitrile hydratase activity, amidase activity, and biomass results are provided in Table 3 below, where the activity is given in units / mg cdw (cell dry weight). One unit of nitrile hydratase activity is related to the ability to convert 1 μmol of acrylonitrile to its corresponding amide per milligram of cells (dry weight) per minute at pH 7.0 and a temperature of 30 ° C. One unit of amidase activity is related to its ability to convert 1 μmol of acrylamide to its corresponding acid per milligram of cells (dry weight) per minute at pH 7.0 and a temperature of 30 ° C. Biomass is recorded as cells filled with g / l cww (cell wet weight).

B. Basically, as described in Example 3A above, the medium was changed to the medium described below, and the enzyme activity and biomass were evaluated by Rhodococcus sp. DAP 96 25 3 cells. More specifically, YEMEA, dextrose, or maltose was added to a modified R3A medium that further contained Hy-Cotton 7803® as a substitute for proteose peptone # 3. The addition of 0.2M asparagine solution started at t = 8 hours and was made at a continuous rate of 120 μl / min. At the end of the fermentation operation, nitrile specific nitrile hydratase activity, amidase activity, and biomass were measured. The results are summarized in Table 4. The resulting increase in biomass was observed upon addition of YEMEA, dextrose, or maltose to the medium.

C. Rhodococcus rhodochrous DAP 96622 cells from a solid medium were used as inoculum for a 20 liter fermentation operation (see Example 2 for details of the fermentation process). The addition of 0.2M asparagine solution started at t = 24 hours and was performed semi-continuously every 6 hours at a rate of 2 ml / min for 50-70 minutes. Hy-Cotton 7803® was used in place of proteose peptone # 3 in the modified R3A medium. At the end of the fermentation operation, nitrile specific nitrile hydratase activity, amidase activity, and biomass were measured. The results are summarized in Table 5.

D. Rhodococcus rhodochrous DAP 96622 cells from the solid medium were used as inoculum for a 20 liter fermentation run. The addition of 0.2M asparagine solution started at t = 12 hours and was performed semi-continuously every 6 hours for 12-85 minutes at a rate of 2.5 ml / min. Cotton Seed Hydrate was used in place of the modified R3A medium proteose peptone # 3. At the end of the fermentation operation, acrylonitrile-specific nitrile hydratase activity, amidase activity, and biomass were measured and the results are summarized in Table 6.

E. Pre-frozen Rhodococcus sp. DAP 96253 cells were used as inoculum for a 20 liter fermentation operation. YEMEA, dextrose, or maltose was added to a modified R3A medium that further contained Hy-Cotton 7803® as a substitute for proteose peptone # 3. The addition of the 0.15 M asparagine solution started at t = 8 hours and was made at a continuous rate of 120 μl / min. At the end of the fermentation operation, nitrile specific nitrile hydratase activity, amidase activity, and biomass were measured. The results are summarized in Table 7.

F. Rhodococcus sp. DAP 96253 cells grown on a three-phase medium were used as inoculum for a 20 liter fermentation operation. A modified R3A medium supplemented by the addition of carbohydrates (ie, YEMEA, dextrose, or maltose) and further containing Cottoned Hydrate instead of proteose peptone # 3 was used. The addition of the 0.15 M asparagine solution started at t = 10 hours and was performed at a continuous rate of 1000 μl / min. At the end of the fermentation operation, nitrile specific nitrile hydratase activity, amidase activity, asparaginase I activity, and biomass were measured. The results are summarized in Table 8.

G. Rhodococcus sp. DAP 96253 cells grown on a biphasic medium were used as inoculum for a 20 liter fermentation operation. Modified R3A medium containing maltose (in place of dextrose) and Hy-Cotton 7803® as a substitute for proteose peptone # 3 was used. The addition of the 0.15 M asparagine solution started at t = 8 hours and was made at a continuous rate of 476 μl / min. At the end of the fermentation operation, acrylonitrile-specific nitrile hydratase activity, amidase activity, and biomass were measured and the results are summarized in Table 9.

Example 4: Immobilization of Rhodococcus seed cells in DEAE cellulose cross-linked with glutaraldehyde US Pat. No. 4,229,536, and Lopez-Gallego et al. (2005) J. Org. Biotechnol. 119: 70-75 is used to fix Rhodococcus species in a matrix containing glutaraldehyde cross-linked DEAE cellulose.
Cell preparation

  Rhodococcus cells are grown in an appropriate medium (eg, YEMEA-maltose + inducing agent, biphasic culture, etc.) and harvested by centrifugation at 8,000 rpm for 10 minutes. The resulting cell pellet is resuspended in 100 ml of 50 mM phosphate buffer (pH 7.2) and centrifuged at 8,000 rpm for 10 minutes. This process of resuspending the cell pellet and centrifuging at 8,000 rpm for 10 minutes is repeated twice. Record the wet wet weight (ww) of the final cell sample. Perform a nitrile hydratase activity on a small sample of cells and evaluate the enzyme activity of the whole cell.

Cell Immobilization The same amount of DEAE cellulose as the harvested Rhodococcus seed cells is obtained, and the cells and DEAE cellulose are resuspended in 100 ml of deionized H ₂ O. A volume of a 25% solution of glutaraldehyde sufficient to achieve a final concentration of 0.5% is added to the cell / DEAE cellulose mixture with stirring. The mixture is stirred for 1 hour, after which 400 ml of deionized H ₂ O is added with further stirring. While stirring, 50% (solution weight) of polyethyleneimine (PEI; MW 750,000) is added. Continue stirring until aggregation is complete. The agglomerated mixture is filtered and extruded with an appropriately sized syringe. Fixed cells are divided into small pieces, dried overnight, and cut into approximately 2-3 mm granules before use.

Example 5: Immobilization of Rhodococcus seed cells in calcium alginate and hardening of calcium alginate beads Methods in Enzymology, Vol. 135 (B) Bucke (1987) “Cell Immobilization in Calcium Alginate” (Academic Press, Inc., San Diego, California, Mosbach, ed), a process adopted in calcium alginate species Used to fix.

Cell Preparation Rhodococcus seed cells are prepared as described in Example 4 above.

Cell Immobilization 25 g of 4% sodium alginate solution is generated by dissolving 1 g sodium alginate in 24 ml 50 mM Tris HCl (pH 7.2). 25 mg of sodium metaperiodate is added to the alginate solution and stirred for 1 hour at 25 ° C. or until the alginate is completely dissolved. Cells prepared as described above are resuspended in 50 mM Tris HCl (pH 7.2) to a final volume of 50 ml and then added to the sodium alginate solution with stirring. The resulting beads are extruded through a 27 gauge needle into 500 ml of 0.1 M CaCl ₂ solution. The needle is generally placed about 2 inches above the solution so that air does not enter the bead and does not penetrate the bead. The beads are cured in CaCl ₂ solution in about 1 hour, then the beads are rinsed with water and stored at 4 ° C. in 0.1 M CaCl ₂ solution before use.

Curing of calcium alginate beads containing Rhodococcus seed cells Calcium alginate beads prepared as outlined above can be further enhanced by crosslinking with PEI. The beads are cultured in 0.5% PEI in ₂ L of 0.1 M CaCl ₂ solution (20 g 50% PEI in 0.1 M CaCl ₂ solution). The pH of the final solution is adjusted to 7.0 with HCl or NaOH as needed and the beads are incubated for 24 hours. The beads are then rinsed with water and stored at 4 ° C. in 0.1 M CaCl ₂ solution before use.

  Many variations and other embodiments of the invention described herein will occur to those skilled in the art to which the invention pertains having the benefit of what has been shown in the above description. Accordingly, it is to be understood that the invention is not limited to the specific embodiments disclosed, and that variations and other embodiments are intended to be included within the scope of the appended claims. I want. Although specific terms have been used herein, they are commonly used and are for the purpose of description only and are not intended to be limiting.

Claims (62)

A method for delaying a plant development process associated with ethylene biosynthesis comprising exposing a plant or plant part to one or more bacteria, the one or more bacteria comprising a nitrile hydratase, an amidase, Producing one or more enzymes selected from the group consisting of asparaginase, and mixtures thereof, wherein the one or more bacteria are selected from the group consisting of Rhodococcus species, Brevibacterium-ketoglutamicum, and mixtures thereof; The method wherein the one or more bacteria are exposed to the plant or plant part in an amount sufficient to delay the plant development process.   The method of claim 1, wherein the one or more bacteria comprises a Rhodococcus species. The method of claim 2, wherein the Rhodococcus sp. Comprises Rhodococcus strain Rhodochrous DAP 96622 , Rhodococcus sp. Strain DAP 96253 , Rhodococcus erythropolis, or a mixture thereof. The one or more bacteria are selected from the group consisting of nitrile hydratase, amidase, and asparaginase, and mixtures thereof, selected from the group consisting of asparagine, glutamine, cobalt, urea, and mixtures thereof. 2. The method of claim 1, wherein said method is induced by exposure to an inducing agent that induces production or increased production of said enzyme .   5. The method of claim 4, wherein the one or more bacteria are induced by exposure to asparagine.   The method of claim 4, wherein the one or more bacteria are induced by exposure to asparagine, cobalt, and urea.   The method of claim 1, wherein the plant or plant part is indirectly exposed to the one or more bacteria.   The method of claim 1, wherein the plant or plant part is directly exposed to the one or more bacteria.   The method of claim 1, wherein the plant development process is fruit or vegetable ripening.   The method according to claim 9, wherein the plant part is a fruit or a vegetable.   The method according to claim 10, wherein the fruit is a climacteric fruit.   12. The method of claim 11, wherein the climacteric fruit is selected from the group consisting of banana, peach, plum, nectarine, apple, tomato, pear, and avocado.   The method according to claim 10, wherein the fruit is a non-climacteric fruit.   The method of claim 9, wherein the plant part is cucumber.   The method of claim 1, wherein the plant part is a flower and the plant development process is flower senescence, wilting, deciduous or petal closure.   16. The method of claim 15, wherein the flower is carnation, rose, orchid, purslane, aoi, or begonia.   The method of claim 1, wherein the plant growth process is leaf litter.   The method of claim 1, wherein the plant part is a cut flower.   The method of claim 1, wherein delaying the plant development process results in an increase in shelf life of the plant or plant part or facilitates long-range transport of the plant or plant part.   The one or more bacteria are fixed and placed in, placed on, or attached to a physical structure suitable for transport or storage of the plant or plant part. the method of. Delaying the plant development process associated with ethylene biosynthesis comprising a catalyst comprising one or more bacteria producing one or more enzymes selected from the group consisting of nitrile hydratase, amidase, asparaginase, and mixtures thereof Wherein the bacteria are selected from the group consisting of Rhodococcus species, Brevibacterium ketoglutamicum, and mixtures thereof, wherein the one or more bacteria are sufficient to delay the plant development process. Equipment provided in quantity.   The apparatus of claim 21, wherein the one or more bacteria comprises a Rhodococcus species. 23. The apparatus of claim 22, wherein the Rhodococcus species comprises Rhodococcus strain Rhodochrous DAP 96622 , Rhodococcus species DAP 96253 , Rhodococcus erythropolis, or mixtures thereof. The one or more bacteria are selected from the group consisting of nitrile hydratase, amidase, and asparaginase, and mixtures thereof, selected from the group consisting of asparagine, glutamine, cobalt, urea, and mixtures thereof. 23. The device of claim 21, wherein said device is induced by exposure to an inducing agent that induces production or increased production of said enzyme .   24. The apparatus of claim 21, wherein the plant development process is selected from the group consisting of fruit or vegetable ripening, flower senescence, wilting, petal closure, and litter.   The one or more bacteria are immobilized within a matrix comprising crosslinked DEAE cellulose, a matrix comprising alginic acid, a matrix comprising carrageen, a matrix comprising crosslinked alginic acid, a matrix comprising crosslinked carrageen, a matrix comprising polyacrylamide, or a calcium alginate bead The device of claim 21.   27. The apparatus of claim 26, wherein the matrix comprises cross-linked DEAE cellulose, and the DEAE cellulose is cross-linked with glutaraldehyde.   23. The apparatus of claim 21, wherein the catalyst is in a catalyst module that is disposed within, disposed on, or attached to a physical structure.   The apparatus of claim 21, further comprising a control device to adjust exposure of the catalyst to a plant or plant part.   The apparatus of claim 21, further comprising a monitoring device for monitoring the effectiveness of the catalyst in delaying the plant growth process.   30. The apparatus of claim 28, wherein the physical structure is selected from the group consisting of a film, a sheet, a covering layer, a slotted chamber, a box, a pouch, and a bag.   30. The apparatus of claim 28, wherein the catalyst module can be removed and replaced with a second catalyst module.   30. The apparatus of claim 28, wherein two or more catalyst modules are disposed within, disposed on, or attached to the physical structure.   29. The apparatus of claim 28, wherein the physical structure allows air flow into the catalyst module.   35. The apparatus of claim 34, further comprising an element for controlling the air flow into the catalyst module.   30. The apparatus of claim 28, wherein the physical structure is provided as a refrigerated structure.   30. The apparatus of claim 28, further comprising an element for controlling a moisture level in the physical structure.   30. The apparatus of claim 28, further comprising an element for adjusting carbon dioxide levels in the physical structure. A breathable catalytic device for delaying plant development processes associated with ethylene biosynthesis ,
A first layer;
A second layer comprising a catalyst comprising one or more bacteria producing one or more enzymes selected from the group consisting of nitrile hydratase, amidase, asparaginase, and mixtures thereof , wherein the bacteria are Rhodococcus A second layer selected from the group consisting of a species, Brevibacterium ketoglutamicum, and mixtures thereof, wherein the one or more bacteria are provided in an amount sufficient to retard the plant development process;
A breathable catalytic device, wherein the first layer provides structural integrity to the device.   40. The device of claim 39, wherein the one or more bacteria comprises a Rhodococcus species. 40. The apparatus of claim 39, wherein the Rhodococcus species comprises Rhodococcus strain Rhodochrous DAP 96622 , Rhodococcus species DAP 96253 , Rhodococcus erythropolis, or mixtures thereof. The one or more bacteria are selected from the group consisting of nitrile hydratase, amidase, and asparaginase, and mixtures thereof, selected from the group consisting of asparagine, glutamine, cobalt, urea, and mixtures thereof. 40. The device of claim 39, wherein said device is induced by exposure to an inducing agent that induces production or increased production of said enzyme .   40. The apparatus of claim 39, wherein the plant development process is selected from the group consisting of fruit or vegetable ripening, flower senescence, and litter.   Further comprising a third layer, the third layer being removed from the second layer to expose an adhesive layer that can be used to attach the catalytic device to a separate structure. 37. The catalytic device of claim 36, wherein   45. The catalyst device according to claim 44, wherein the second layer is the pressure-sensitive adhesive layer.   A fourth layer adjacent to the third layer that can be used to attach the catalyst structure to a separate structure and can be removed from the third layer to expose the adhesive layer. The catalyst device according to claim 44, further comprising:   47. The catalyst device according to claim 46, wherein the third layer is the adhesive layer.   40. A breathable bag or pouch comprising the catalyst device of claim 39. The plant or plant part, comprising nitrile hydratase, amidase, asparaginase, and the step of exposing the enzyme extract of one or more bacteria which produce one or more enzymes selected from the group consisting of mixtures thereof, ethylene A method for delaying a plant development process associated with biosynthesis , wherein the bacterium is selected from the group consisting of Rhodococcus species, Brevibacterium ketoglutamicum, and mixtures thereof, wherein the bacterium comprises asparagine, glutamine, Inducing production or increased production of one or more enzymes selected from the group consisting of nitrile hydratase, amidase, and asparaginase, and mixtures thereof selected from the group consisting of cobalt, urea, and mixtures thereof Induced by inducer Wherein the enzyme extract is an amount sufficient to delay the plant development process, it is exposed to the plant or plant portion. A method for delaying a plant development process associated with ethylene biosynthesis comprising exposing a plant or plant part to one or more bacteria, the one or more bacteria comprising a nitrile hydratase, an amidase, Producing one or more enzymes selected from the group consisting of asparaginase, and mixtures thereof, wherein the one or more bacteria are selected from the group consisting of asparagine, glutamine, cobalt, urea, and mixtures thereof ; Pseudomonas chlorolafis induced by exposure to an inducer that induces production or increased production of one or more enzymes selected from the group consisting of nitrile hydratase, amidase, and asparaginase, and mixtures thereof The one or more bacteria are the plant growth process. In an amount sufficient to delay processes, it is exposed to the plant or plant portion.   51. The method of claim 50, wherein the one or more bacteria are induced by exposure to asparagine or asparagine, cobalt, and urea.   51. The method of claim 50, wherein the plant or plant part is exposed directly or indirectly to the one or more bacteria.   51. The method of claim 50, wherein the plant development process is fruit or vegetable ripening.   51. The method of claim 50, wherein the plant part is a fruit, vegetable, or flower.   55. The method of claim 54, wherein the fruit is a non-climacteric or climacteric fruit.   56. The method of claim 55, wherein the climacteric fruit is selected from the group consisting of banana, pear, plum, nectarine, apple, tomato, peach, and avocado.   51. The method of claim 50, wherein the plant part is a flower and the plant development process is flower senescence, wilting, deciduous or petal closure.   58. The method of claim 57, wherein the flower is carnation, rose, orchid, purslane, mallow, or begonia.   51. The method of claim 50, wherein the plant development process is leaf litter.   51. The method of claim 50, wherein the plant part is a cut flower.   51. The method of claim 50, wherein delaying the plant development process results in an increase in shelf life of the plant or plant part or facilitates long-range transport of the plant or plant part.   51. The one or more bacteria are fixed and placed in, placed on, or attached to a physical structure suitable for transport or storage of the plant or plant part. the method of.

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